Simultaneous fluorescence imaging of hydrogen peroxide in mitochondria and endoplasmic reticulum during apoptosis

Cell apoptosis is a biochemical and molecular pathway essential for maintaining cellular homeostasis. It is an integrated process involving in a series of signal transduction cascades. Moreover, the apoptotic pathways may be initiated inside various subcellular organelles. Increasing evidence indicates that hydrogen peroxide (H(2)O(2)) is closely related to cell apoptosis, particularly in the mitochondria. However, during the apoptotic process, the synergetic variation of H(2)O(2) levels in different compartments is seldom explored, particularly in two important organelles: mitochondria and endoplasmic reticulum (ER). To solve this problem, we developed two new organelle-specific fluorescent probes termed MI-H(2)O(2) and ER-H(2)O(2) that can detect H(2)O(2) in mitochondria and ER, respectively or simultaneously. Experimental results demonstrated that MI-H(2)O(2) and ER-H(2)O(2) display distinguishable excitation and emission spectra, as well as excellent organelle targeting capabilities. Therefore, we used MI-H(2)O(2) and ER-H(2)O(2) to successfully image exogenous or endogenous hydrogen peroxide in the mitochondria and ER. Interestingly, during diverse apoptotic stimuli, dual-color fluorescence imaging results revealed that the changes of H(2)O(2) levels in mitochondria and ER are different. The H(2)O(2) levels are enhanced in both the mitochondria and ER during the l-buthionine sulfoximine (BSO)-treated cell apoptosis process. During mitochondria-oriented apoptosis induced by carbonyl cyanide m-chlorophenylhydrazone (CCCP) or rotenone, H(2)O(2) levels prominently and continuously increase in the mitochondria, whereas the ER H(2)O(2) levels were found to rise subsequently after a delay. Moreover, during ER-oriented apoptosis induced by tunicamycin, ER is the major site for overproduction of H(2)O(2), and delayed elevation of the H(2)O(2) levels was found in the mitochondria. Altogether, this dual-probe and multicolor imaging approach may offer a proven methodology for studying molecular communication events on H(2)O(2)-related apoptosis and also other physiological and pathological processes within different subcellular organelles.